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SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex.

Matunis MJ, Wu J, Blobel G - J. Cell Biol. (1998)

Bottom Line: SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four.Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal.This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York 10021, USA. Matunim@rockvax.rockefeller.edu

ABSTRACT
RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

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RanGAP1/PK fusion proteins are SUMO-1  modified in vivo. Cells transfected as in Fig. 4 were lysed  in SDS sample buffer and analyzed by immunoblot analysis with the anti-myc monoclonal antibody, 9E10. In  cells transfected with native  pyruvate kinase, a single specific band migrating at 50 kD  is detected (lane 1). The  band migrating at 65 kD is  nonspecific and detected in  cells transfected with no  DNA (lane 5). Two forms of  the fusion proteins NΔ419/ PK (lane 2), NΔ470/PK (lane  3), and NΔ502/PK (lane 4)  are detected, the lower molecular mass forms corresponding  to unmodified protein, the  higher molecular mass forms corresponding to SUMO-1–modified  protein. Molecular mass standards are indicated on the left.
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Figure 5: RanGAP1/PK fusion proteins are SUMO-1 modified in vivo. Cells transfected as in Fig. 4 were lysed in SDS sample buffer and analyzed by immunoblot analysis with the anti-myc monoclonal antibody, 9E10. In cells transfected with native pyruvate kinase, a single specific band migrating at 50 kD is detected (lane 1). The band migrating at 65 kD is nonspecific and detected in cells transfected with no DNA (lane 5). Two forms of the fusion proteins NΔ419/ PK (lane 2), NΔ470/PK (lane 3), and NΔ502/PK (lane 4) are detected, the lower molecular mass forms corresponding to unmodified protein, the higher molecular mass forms corresponding to SUMO-1–modified protein. Molecular mass standards are indicated on the left.

Mentions: To determine whether the proteins transiently expressed in vivo were modified as expected, lysates from transfected cells were analyzed by immunoblot analysis using an antibody to the myc epitope tag. In cells expressing NΔ419/PK and NΔ470/PK, SUMO-1–modified forms of the proteins were detected, with approximately half of the proteins present in the modified form (Fig. 5, lanes 2 and 3). This ratio of modified to unmodified protein was noticeably higher than that observed with wild-type RanGAP1, where a relatively small amount of modified protein was detected (see Fig. 2, lane 1). Surprisingly, NΔ502/PK was also modified in vivo, contrary to the in vitro translation result, with the ratio of modified to unmodified forms again being ∼50% (Fig. 5, lane 4). SUMO-1 modification of the transiently expressed fusion proteins was confirmed by immunoblot analysis with an antibody specific for SUMO-1 (data not shown).


SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex.

Matunis MJ, Wu J, Blobel G - J. Cell Biol. (1998)

RanGAP1/PK fusion proteins are SUMO-1  modified in vivo. Cells transfected as in Fig. 4 were lysed  in SDS sample buffer and analyzed by immunoblot analysis with the anti-myc monoclonal antibody, 9E10. In  cells transfected with native  pyruvate kinase, a single specific band migrating at 50 kD  is detected (lane 1). The  band migrating at 65 kD is  nonspecific and detected in  cells transfected with no  DNA (lane 5). Two forms of  the fusion proteins NΔ419/ PK (lane 2), NΔ470/PK (lane  3), and NΔ502/PK (lane 4)  are detected, the lower molecular mass forms corresponding  to unmodified protein, the  higher molecular mass forms corresponding to SUMO-1–modified  protein. Molecular mass standards are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140169&req=5

Figure 5: RanGAP1/PK fusion proteins are SUMO-1 modified in vivo. Cells transfected as in Fig. 4 were lysed in SDS sample buffer and analyzed by immunoblot analysis with the anti-myc monoclonal antibody, 9E10. In cells transfected with native pyruvate kinase, a single specific band migrating at 50 kD is detected (lane 1). The band migrating at 65 kD is nonspecific and detected in cells transfected with no DNA (lane 5). Two forms of the fusion proteins NΔ419/ PK (lane 2), NΔ470/PK (lane 3), and NΔ502/PK (lane 4) are detected, the lower molecular mass forms corresponding to unmodified protein, the higher molecular mass forms corresponding to SUMO-1–modified protein. Molecular mass standards are indicated on the left.
Mentions: To determine whether the proteins transiently expressed in vivo were modified as expected, lysates from transfected cells were analyzed by immunoblot analysis using an antibody to the myc epitope tag. In cells expressing NΔ419/PK and NΔ470/PK, SUMO-1–modified forms of the proteins were detected, with approximately half of the proteins present in the modified form (Fig. 5, lanes 2 and 3). This ratio of modified to unmodified protein was noticeably higher than that observed with wild-type RanGAP1, where a relatively small amount of modified protein was detected (see Fig. 2, lane 1). Surprisingly, NΔ502/PK was also modified in vivo, contrary to the in vitro translation result, with the ratio of modified to unmodified forms again being ∼50% (Fig. 5, lane 4). SUMO-1 modification of the transiently expressed fusion proteins was confirmed by immunoblot analysis with an antibody specific for SUMO-1 (data not shown).

Bottom Line: SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four.Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal.This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York 10021, USA. Matunim@rockvax.rockefeller.edu

ABSTRACT
RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

Show MeSH
Related in: MedlinePlus